Heparan sulfate (HS) is a polysaccharide that constitutes a key component of the extracellular matrix (EM) in all animals. HS has been shown to bind ligands and their receptors by way of complex modifications of the sugar residues, including sulfations and epimerizations. HS can protect proteins from degradation, chaperone a ligand through the EM, set up ligand gradients, and directly interact with both the receptor and ligand. HS modifications have been shown to be crucial for correct neuronal development in both vertebrates and invertebrates such as drosophila and C. elegans. These studies have led to the hypothesis of a HS code that provides cells with the ability to modulate extracellular interactions in their local environment. This hypothesis posits that metazoans harbor a diverse HS sugar landscape defined by complex modification patterns that modulate protein functions (e.g., of receptors and ligands) through direct interactions with the sugars. It is unknown whether such a sugar landscape exists and which processes it may influence. One challenge is that specifically modified sugars cannot be visualized in living animals. Using C. elegans as a model, I have developed a method that allows me to visualize specifically modified HS in vivo. This method is based on transgenic expression of engineered HS-specific single chain variable fragment antibodies (scFv). My preliminary data, which use four scFvs with distinct binding specificities of HS in vitro, show that I can, first, label distinctly modified HS motifs and, secod, reveal that diverse HS modification patterns are associated with defined structures of the nervous system. In my first specific aim, I will further characterize these scFv antibodies and describe the anatomical structures they bind. This will allow me to define the HS sugar landscape of a living multicellular organism for the first time. In my second specific aim, I will determine the function of HS motifs in synaptic transmission. Specifically, I will use synaptic function assays and genetic epistasis experiments to determine how HS motifs interact with cholinergic synaptic function. In summary, this project aims to visualize the HS landscape and determine how HS motifs function in cholinergic synaptic transmission to better understand the structure-function relationships of defined HS motifs in vivo.